PROJECT SUMMARY The development and commercialization of microfluidic-based single cell RNA-seq platforms has enabled the transcriptomes of thousands of single cells to be profiled at a time, allowing researchers to differentiate between cell types and study cell fate decisions. However, none of these new technologies can correlate single cell genetic or epigentic variations with gene expression at high throughput. Fundamentally, current microfluidic technologies are either too inflexible to perform multiple types of sequencing on the same single cell, or are too low throughput to address many biological questions. We will overcome this limitation by utilizing a new microfluidic technology, Printed Droplet Microfluidics, to build a platform to perform high throughput single cell multi-omics. Printed Droplet Microfluidics utilizes a unique droplet microfluidic print head that serves as a deterministic single cell and droplet printer, and allows nanoliter-scale fluidic manipulations to be performed in an array format, including multiple protocols on the same single cell lysate. In this project, we will build a platform to perform chromatin accessibility (ATAC-seq) and transcriptome sequencing on 1000 single cells. In Specific Aim 1, we will adapt a single cell RNA-seq protocol to the printed droplet array, and develop methods to identify the original geographic location of a cell in the RNA-seq data. In Specific Aim 2, we will develop a high throughput single cell ATAC-seq protocol in an array format, with methods again being developed to positionally label single cell ATAC-seq data. Achievement of these aims will prove that multiple single cell sample preparation protocols can be linked together and performed on the same single cell lysate. A proposed phase II project would involve full integration RNA-seq and ATAC-seq protocols, scaling throughput > 10,000 single cells, and early productization of the hardware and disposables.